Triptolide Induces Apoptosis Of Human Jurkat And K562Leukemia Cells Via Inhibiting Transcription Of HERV-K Np9

Aims:1. To reveal transcription levels of HERV-K Np9gene in leukemia cell lines and primary leukemia cell samples from patients with various leukemia.2. To assess the effects of triptolide (TPL) on transcription of HERV-K Np9and its downstream molecules in K562and Jurkat leukemia cells.3. To investigate the relationship between TPL-induced apoptosis and TPL-mediated inhibition of Np9gene in K562and Jurkat leukemia cells.Methods:1. Jurkat, K562, K562/ADR, Raji, NB4, KM3, CML-BC, KU812, KG-1, THP-1, Kasumi-1, KCL-22cells were treated with triptolide at concentrations of0.5-64nmol/L for72h and primary leukemia cells were treated with triptolide at concentrations of0.5-64nmol/L for48h, and then collected for analyses of cell viability by MTT assay and the IC50values of triptolide for different cell lines and primary leukemia cell samples.2. Semi-quantative RT-PCR was used to detect the Np9mRNA level in12various human hematopoietic malignant cell lines and primary leukemia cell.3. Flow cytometry was used to analyze apoptosis levels of Jurkat and K562cells after treatment with triptolide at concentrations of0-16nmol/L for48h.4. Semi-quantative RT-PCR and Kodak ID3.6were used to analyze mRNA levels of Np9gene in Jurkat, K562cells after treatment with TPL.5. Correlation between TPL-mediated inhibition of Np9transcription and TPL-induced apoptosis of leukemia cells was analyzed by SPSS19.0software.6. Western blot was employed to determine Np9downstream signaling molecules c-myc,β-catenin, p-ERK, p-AKT and Notchl protein level in Jurkat and K562cells after exposure to triptolide at various concentrations for48h.7. The expression levels of activated PARP and activated caspase-3in Jurkat and K562cells were determined by western blot after treatment with triptolide.Results:1. Triptolide potently inhibited the proliferation of Jurkat, K562, K562/ADR, Raji, NB4, KM3, CML-BC, KU812, KG-1, THP-1, Kasumi-1and KCL-22in dose-dependent manner, and their IC50values were ranged from1.4nmol/L to27.1nmol/L. Triptolide also exhibited strong inhibition for5primary leukemia cell samples, and their IC50values were ranged from9.13nmol/L to29.2nmol/L.2. Out of12various human hematopoietic malignant cell lines, Jurkat, K652, K652/ADR, KM3cells and primary leukemia cells expressed high levels of Np9mRNA.3. Triptolide induced apoptosis of Jurkat and K562cells in dose-dependent manner.4. Triptolide decreased Np9mRNA transcription level in Jurakt and K562cells as well as primary human leukemia samples in dose-dependent manner.5.There was a significant correlation between TPL-mediated inhibition of Np9 transcription and TPL-induced apoptosis in Jurkat cells (R2=0.907, p<0.05) and K562cells (R2=0.987, p<0.05).6. Triptolide inhibited Np9mRNA with a concomitant decrease of its downstream molecules c-myc, β-catenin, p-ERK, p-AKT and Notch1protein levels in Jurakt and K562cells.7. Triptolide increased levels of activated PARP and activated caspase-3protein level in a dose-dependent manner.Conclusion:1. Triptolide potently inhibits the proliferation of a variety of hematopoietic tumor cell lines as well as primary leukemia cells in a dose-dependent manner.2. HERV-K Np9mRNA is highly expressed in several hematopoietic tumor cell lines as well as primary leukemia cells.3. There was a significant correlation between TPL-mediated inhibition of Np9transcription and TPL-induced apoptosis in leukemia cells, suggesting that TPL-mediated inhibition of Np9mRNA is one of the important mechanisms by which TPL induces apoptosis of leukemia cells.4. Triptolide inhibits Np9mRNA with a concomitant decreasing of its downstream molecules c-myc, β-catenin, p-ERK, p-AKT and Notch1protein levels in Jurakt and K562cells.5. HERV-K Np9might be a novel therapeutic target for leukemia, and triptolide might be a novel transcription inhibitor of HERV-K Np9gene.